CN111763703A - A kind of method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent - Google Patents

Abstract

The invention discloses a method for enzymatically synthesizing sucrose-6-ethyl ester in an organic solvent. The method comprises the following steps: adding sucrose into an organic solvent, then adding immobilized lipase and vinyl acetate, mixing uniformly, and at 30- 40°C water bath shaker rotation speed 120-180r/min, reaction time 12-20h, filter, filter cake is recovered immobilized lipase, filtrate is vacuum distilled to recover solvent to obtain sucrose-6-ethyl ester product. The present invention adopts N,N-dimethylformamide (DMF) as the main solvent for enzymatically synthesizing sucrose-6-ethyl ester, which is compounded with tert-amyl alcohol or tert-butanol, and compared with other organic solvents, it has better enzyme activity retention. Good, the solubility of sucrose can reach 10%, the esterification rate of sucrose is as high as more than 85%, the proportion of sucrose-6-ethyl ester in the total ester is more than 85%, the viscosity of the synthetic reaction solvent system is small, and the advantages of easy recovery.

Description

A kind of method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent

(1) Technical field

The invention relates to a method for enzymatically synthesizing sucrose-6-ethyl ester in an organic solvent.

(2) Background technology

At home and abroad, sucrose-6-ethyl ester is chemically synthesized in N,N-dimethylformamide (DMF) solvent. Sucrose is dissolved in DMF solvent and undergoes esterification with trimethyl orthoacetate. The esterification reaction occurs under the catalysis of tin oxide. The chemical method has a great impact on the environment, and the lipase-catalyzed sucrose esterification is a green manufacturing and has an ideal development prospect.

Sucrose-6-ethyl ester is a key intermediate in the production of non-calorie high-intensity sweetener sucralose, so the green and safe lipase-catalyzed synthesis of sucrose-6-ethyl ester technology has carried out a lot of research and development at home and abroad. In order to make sucrose dissolve better and lipase can play catalytic activity at the same time, the reported studies use organic solvent mixed with water as the reaction solvent, or ionic liquid as the reaction solvent, but the sucrose esterification rate is below 70%. . Using dimethyl sulfoxide compound medium chain alcohol as the solvent of sucrose esterification reaction, the effect of lipase catalyzing synthesis of sucrose-6-ethyl ester is better. However, dimethyl sulfoxide has a serious impact on the enzyme activity, the solubility of sucrose in dimethyl sulfoxide is not large enough, and the viscosity of dimethyl sulfoxide is relatively large, which increases the difficulty of operation in the reaction process. To this end, research on the ideal organic solvent for enzymatic synthesis of sucrose-6-ethyl ester has become a hot spot of technological invention at home and abroad.

(3) Contents of the invention

The purpose of the present invention is to improve the esterification efficiency of the enzymatic synthesis of sucrose-6-ethyl ester in an organic solvent, and to provide a method for the enzymatic synthesis of sucrose-6-ethyl ester in an organic solvent, which can replace the current chemical synthesis of sucrose-6-ethyl ester. The ethyl ester process, applied to the production of non-calorie high-intensity sweetener sucralose, can greatly reduce the impact of the production process on the environment and realize the green transformation of sucralose production.

The technical scheme adopted in the present invention is:

The invention provides a method for enzymatically synthesizing sucrose-6-ethyl ester in an organic solvent. The method comprises the following steps: adding sucrose into an organic solvent, then adding immobilized lipase and vinyl acetate, mixing uniformly, and heating the sucrose at 30-40 ℃ water bath and shaker rotating speed of 120-180r/min for 12-20h (preferably 30℃, 160r/min reaction for 16h), filtered, the filter cake is the recovered immobilized lipase, and the filtrate is vacuum distilled to recover the solvent to obtain sucrose-6 -Ethyl ester products (sucrose ester content is more than 85%, and sucrose-6-ethyl ester accounts for more than 85% of total esters); the organic solvent is composed of N,N-dimethylformamide and tert-amyl alcohol or tert-butanol. The volume concentration is 55-30%: 45-70% is mixed, and the total amount is 100%; the immobilized lipase is formed by adsorbing and immobilizing the lipase with a macroporous resin.

Further, the organic solvent is prepared by mixing N,N-dimethylformamide and tert-amyl alcohol with a volume concentration of 55-30%: 45-70%, and the total amount is 100%.

Further, the volume addition amount of the organic solvent is 125-330ml/g (preferably 160-250ml/g) in terms of the weight of sucrose; the mass ratio of the sucrose to the immobilized lipase is 1:0.2-1.0 (preferably 1:0.5- 0.7); the mass ratio of sucrose and vinyl acetate is 1:1-5 (preferably 1:2-4).

Further, the immobilized lipase is prepared as follows: the lipase is dissolved in deionized water (stirring makes the enzyme dissolve, and the insoluble component is then removed by centrifugation, and the enzyme concentration in the enzyme solution is controlled at 2-4mg/ml, adding twelve Hydrate disodium hydrogen phosphate and sodium dihydrogen phosphate, stir evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer, add macroporous resin, keep at 20-35 ℃, and stir at 80-100 r/min , adsorbed for 1-3h (preferably at 30°C, 90 rpm, adsorbed for 2h), filtered, and vacuum-dried the filter cake (preferably at 40°C) to a water content of 3-5% (preferably 4.0%) to obtain immobilized lipase; Described deionized water volume consumption is 150-500ml/g (preferably 160-250ml/g) by lipase weight; Described lipase and dodecahydrate disodium hydrogen phosphate and sodium dihydrogen phosphate mass ratio are respectively 1:0.5 -1.5 (preferably 1:0.7-1.1) and 1:0.5-1.0 (preferably 1:0.5-0.8); the weight ratio of the lipase to the macroporous resin is 1:2-4. The macroporous resin type includes D4006 Type, D3520, D1300.

The macroporous resin is pretreated before adding the enzyme solution, and the pretreatment method is as follows: adding 10 times the mass of the macroporous resin into a 95% ethanol aqueous solution with a volume concentration of 95%, at room temperature (25-30° C.), 150 r/min Under the condition of shaking and washing for 24 hours, and replacing the 95% ethanol aqueous solution twice in the middle, after shaking and washing, wash five times with twice the mass of distilled water each time, suction filtration, and the filter cake is the macroporous resin that has completed the pretreatment.

The enzymatic activity of the lipase is 100,000-300,000 units/gram, preferably Aspergillus niger lipase or Candida lipase.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention adopts N,N-dimethylformamide (DMF) as the main solvent for the enzymatic synthesis of sucrose-6-ethyl ester, and is combined with tertiary amyl alcohol or tertiary ethyl alcohol. Butanol compound, compared with other organic solvents, has better enzymatic activity retention, sucrose solubility can reach 10%, sucrose esterification rate is as high as 85%, and sucrose-6-ethyl ester accounts for more than 85% of total esters. The synthesis reaction The solvent system has the advantages of low viscosity and easy recovery. The solvent has little effect on the enzymatic activity, has suitable viscosity and boiling point, and is suitable for the reaction operation of enzymatic synthesis of sucrose-6-ethyl ester.

(4) Description of drawings

Figure 1. Standard curve of bovine serum albumin.

2 is a liquid chromatogram of sucrose-6-ethyl ester prepared in Example 1.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

In the embodiment of the present invention, sucrose was purchased from Guangdong Chemical Reagent Engineering Technology Research and Development Center (chemically pure), and N,N-dimethylformamide and tert-amyl alcohol were purchased from Sinopharm Chemical Reagent Co., Ltd. (chemically pure); acetic acid Vinyl ester was purchased from Jiangsu Yonghua Fine Chemicals Co., Ltd. (chemically pure); the room temperature in the present invention refers to 25-30°C.

Example 1:

1. Immobilization of lipase

Preparation of 0.05mol/L pH7.4 phosphate buffer: 8.7 g of disodium hydrogen phosphate dodecahydrate and 6.1 g of sodium dihydrogen phosphate, dissolved in 2000 ml of deionized water.

Macroporous resin pretreatment: D4006 type macroporous resin (purchased from Tianjin Haoju Resin Technology Co., Ltd.) was added with a volume concentration of 10 times the mass of 95% ethanol aqueous solution, and shaken and washed for 24 hours at room temperature and 150 r/min. The 95% ethanol aqueous solution was replaced twice in the middle, and after the shaking, washed five times with twice the mass of distilled water each time, suction filtered, and the filter cake was the pretreated D4006 macroporous resin for use.

Dissolve 20 grams of Aspergillus niger lipase (Aspergillus niger lipase, produced by Shenzhen Lvweikang Biological Engineering Co., Ltd., 300,000 units/g) in 5000 ml of deionized water, stir to dissolve the enzyme, and filter to remove insoluble components. Then 21.8 g of disodium hydrogen phosphate dodecahydrate and 15.3 g of sodium dihydrogen phosphate were added, and stirred evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer. Add 80 grams of pretreated D4006 macroporous resin, keep at 30 °C, stir at 90 rpm, absorb for 2 h, filter, detect the lipase content in the filtrate, and calculate the immobilization rate of 94.3%; filter cake at 40 °C under vacuum It was dried to a water content of 4.7% to obtain 57.8 g of immobilized lipase.

The content of lipase was determined by Coomassie brilliant blue spectrophotometry, and the color was developed with Coomassie brilliant blue reagent and enzyme protein solution, and the absorbance was measured at 595 nm. Using bovine serum albumin as the standard substance to make the standard curve, the enzyme protein content was obtained by the standard curve method. After the macroporous resin adsorbs lipase, measure the amount of enzyme protein in the filtrate after filtration and separation of the resin, according to: immobilization rate % = (1- amount of enzyme protein in filtrate / amount of enzyme protein in enzyme solution) × 100%, Calculate the immobilization rate of resin-immobilized lipase, the specific method is as follows:

Accurately weigh 100 mg of Coomassie brilliant blue G-250, dissolve it in 50 mL of 95% ethanol aqueous solution by volume, add 100 mL of 85% phosphoric acid aqueous solution by volume, and dilute to 1000 mL with distilled water to prepare Coomassie brilliant blue reagent. Prepare a standard solution of bovine serum albumin (BSA), measure the absorbance at 595 nm, and make a standard curve according to Table 1 (Figure 1). The absorbance of the solution was measured at 595 nm, and the absorbance A was used as the ordinate and the protein content as the abscissa. The standard curve is shown in Figure 1.

It can be seen from Fig. 1 that the standard curve equation is Y=6.3014X-0.0062, and R ² =0.9948.

Table 1 Preparation of standard curve of bovine serum albumin

Calculation of immobilization rate: Measure the absorbance values A ₁ and A ₂ of the initial enzyme solution and the enzyme solution after resin adsorption, calculate the protein content X ₁ and X ₂ according to the standard curve, and then calculate the total protein content b ₁ and b ₂ .

b (mg) = protein content (X) / sampling volume (ml) × volume of enzyme solution (mL)

The formula for calculating the immobilization rate is as follows: Immobilization rate %=(b ₁ -b ₂ )/b ₁ ×100%

2. Synthesis of sucrose-6-ethyl ester

N,N-dimethylformamide and tert-amyl alcohol are mixed into 1000ml of compound solvent at a volume concentration of 55%:45%, add 100 grams of sucrose, stir well to completely dissolve the sucrose in the compound solvent, add the prepared solvent in step 1 60 grams of immobilized lipase, then add 250 grams of vinyl acetate, mix well, 35 ℃ water bath shaker rotation speed 160r/min, reaction time 20h, filter and isolate the immobilized lipase, and vacuum distillation to recover the solvent to obtain sucrose ethyl esterification Product 98.7g. Into liquid chromatography analysis, each group of three parallel samples. The results of liquid chromatography showed that the esterification rate was 85.5%, and the 6-position esterification selectivity of sucrose was 85.2%.

Liquid chromatography detection method: high performance liquid chromatography HPLC (Agilent 1200Series); the chromatographic column is ZORBAXSB-Aq (5 μm, 4.6×250 mm), and the detector is an evaporative light scattering detector Alltech-ELSD3300. The mobile phase was 7.0% methanol aqueous solution, the flow rate was 0.8 mL/min, the column temperature was 30 °C, the injection volume was 5 μL; the temperature of the evaporative light detector was 85 °C, and the nitrogen flow rate was 1.5 L/min. The sample was appropriately diluted with tert-amyl alcohol, filtered through a 45 μm organic nylon microporous membrane, and then injected into the sample for analysis. The liquid chromatogram was recorded, and the sucrose esterification rate and the mass content of sucrose-6-ethyl ester were calculated by the normalization method.

Esterification rate % = amount of sucrose to produce product sucrose ester / amount of initial sucrose x 100%

Mass content of sucrose-6-ethyl ester (ie selectivity) %=(amount of sucrose-6-acetate/amount of total sucrose ester)×100%.

Example 2:

1. Immobilization of lipase

Dissolve 20 g of Aspergillus niger lipase (purchased from Nanning Pangbo Bioengineering Co., Ltd., 300,000 units/g) in 5000 ml of deionized water, stir to dissolve the enzyme, and filter out insoluble components. 21.8 g of disodium hydrogen phosphate dodecahydrate and 15.3 g of sodium dihydrogen phosphate were added, and stirred evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer. Then add 40 grams of pretreated D3520 macroporous resin (purchased from Nankai University Chemical Factory, the pretreatment method is the same as in Example 1), keep at 30 ° C and keep stirring at 90 rpm for 3h, and filter, using Example 1 Methods The lipase content of the filtrate was detected, and the immobilization rate was calculated to be 91.7%; the filter cake was vacuum-dried at 40°C to a water content of 3.2% to obtain 36.9 g of immobilized lipase.

2. Synthesis of sucrose-6-ethyl ester

Mix N,N-dimethylformamide and tert-amyl alcohol with a volume concentration of 40%: 60% to form 1000ml of a composite solvent, add 65 grams of sucrose, stir well to completely dissolve the sucrose in the composite solvent, and add the fixative prepared in step 1. 35 grams of lipase, then add 200 grams of vinyl acetate, mix well, 30 ℃ water bath shaker rotation speed 160r/min, reaction time 16h, filter and isolate the immobilized lipase, and vacuum distillation to recover the solvent to obtain sucrose ethyl ester product 66.8 g. The method of Example 1 was used for liquid chromatographic analysis, three parallel samples in each group, the esterification rate was 88.3%, and the 6-position esterification selectivity of sucrose was 87.6%.

Example 3:

1. Immobilization of lipase

Dissolve 30 g of Candida rugosa lipase (Candida rugosa lipase, produced by Beijing Kaitai New Century Biotechnology Co., Ltd., 130,000 units/g) in 5000 ml of deionized water, stir to dissolve the enzyme, and filter out insoluble components. Add 21.8 g of disodium hydrogen phosphate dodecahydrate and 15.3 g of sodium dihydrogen phosphate, stir evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer. Then add 90 grams of D1300 type macroporous resin (Tianjin Haoju Resin Technology Co., Ltd.) pretreated by the method of Example 1, keep warm at 30 ° C, under stirring at 90 rpm, adsorb for 2h, filter, and detect the filtrate by the method of Example 1 The lipase content was calculated to obtain an immobilization rate of 93.2%; the filter cake was vacuum-dried at 40° C. to a water content of 4.1% to obtain 73.6 g of immobilized lipase.

2. Synthesis of sucrose-6-ethyl ester

Mix N,N-dimethylformamide and tert-amyl alcohol with a volume concentration of 30%: 70% to form a composite solvent of 1000ml, add 30 grams of sucrose, stir well to completely dissolve the sucrose in the composite solvent; add the fixed solution prepared in step 1. Add 20 grams of lipase, then add 100 grams of vinyl acetate, mix well, 40 ° C water bath shaker speed 160r/min, reaction time 12h, filter and separate the immobilized lipase, and vacuum distillation to recover the solvent to obtain sucrose ethyl ester product 26.6 g. The method of Example 1 was used for liquid chromatographic analysis. There were three parallel samples in each group, the esterification rate was 85.1%, and the 6-position esterification selectivity of sucrose was 85.2%.

Example 4:

1. Immobilization of lipase

Dissolve 20 grams of Aspergillus niger lipase (Aspergillus niger lipase, produced by Nanning Pangbo Biological Engineering Co., Ltd., 300,000 units/g) in 5000 ml of deionized water, stir to dissolve the enzyme, and filter to remove insoluble components. Add 21.8 g of disodium hydrogen phosphate dodecahydrate and 15.3 g of sodium dihydrogen phosphate, stir evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer. Add 50 grams of D3520 macroporous resin (Nankai University Chemical Plant) pretreated by the method of Example 1, keep at 30°C, keep stirring at 90 rpm, absorb for 3h, filter, and detect the lipase content of the filtrate by the method of Example 1, The immobilization rate was calculated to be 92.4%; the filter cake was vacuum-dried at 40° C. to a water content of 3.1% to obtain 41.2 g of immobilized lipase.

2. Synthesis of sucrose-6-ethyl ester

N,N-dimethylformamide and tert-butanol are mixed with a volume concentration of 40%:60% to form 1000ml of a composite solvent, add 65 grams of sucrose, stir well to completely dissolve the sucrose in the composite solvent, and add the immobilized solvent prepared in step 1. 35 grams of lipase, then add 250 grams of vinyl acetate, mix well, 30 ℃ water bath shaker rotation speed 160r/min, reaction time 16h, filter and isolate the immobilized enzyme, and vacuum distillation to recover the solvent to obtain 65.7g of sucrose-ethyl ester product . The method of Example 1 was used for liquid chromatographic analysis. There were three parallel samples in each group, the esterification rate was 85.7%, and the 6-position esterification selectivity of sucrose was 85.2%.

Comparative Example 1:

1. Immobilization of lipase

Dissolve 20 grams of Aspergillus niger lipase (Aspergillus niger lipase, produced by Nanning Pangbo Biological Engineering Co., Ltd., 300,000 units/g) in 5000 ml of deionized water, stir to dissolve the enzyme, and filter to remove insoluble components. 21.8 g of disodium hydrogen phosphate dodecahydrate and 15.3 g of sodium dihydrogen phosphate were added, and stirred evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer. Then add 50 grams of D3520 type macroporous resin (Nankai University Chemical Factory) pretreated by the method of Example 1, keep at 30 ° C, under stirring at 90 rpm, adsorb for 3h, filter, and detect the lipase content of the filtrate by the method of Example 1 , the immobilization rate was calculated to be 92.4%; the filter cake was vacuum-dried at 40° C. to a water content of 3.1% to obtain 41.2 g of immobilized lipase.

2. Synthesis of sucrose-6-ethyl ester

Dimethyl sulfoxide and tert-amyl alcohol are mixed at a volume concentration of 40%: 60% to form a compound solvent of 1000ml, add 65 grams of sucrose, stir well to completely dissolve the sucrose in the compound solvent, and add 40 grams of immobilized lipase prepared in step 1. , then add 250 g of vinyl acetate, mix evenly, 35 ° C water bath shaker speed 160 r/min, reaction time 16 h, filter to separate the immobilized enzyme, and vacuum distillation to recover the solvent to obtain 31.7 g of sucrose ethyl esterification product. The method of Example 1 was used for liquid chromatographic analysis. There were three parallel samples in each group, the esterification rate was 46.7%, and the 6-position esterification selectivity of sucrose was 88.3%.

Comparative Example 2:

1. Immobilization of lipase

Dissolve 20 grams of Aspergillus niger lipase (Aspergillus niger lipase, produced by Nanning Pangbo Biological Engineering Co., Ltd., 300,000 units/g) in 5000 ml of deionized water, stir to dissolve the enzyme, and filter to remove insoluble components. 21.8 g of disodium hydrogen phosphate dodecahydrate and 15.3 g of sodium dihydrogen phosphate were added, and stirred evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer. Add 50 grams of D3520 macroporous resin (Nankai University Chemical Plant) pretreated by the method of Example 1, keep at 30°C, keep stirring at 90 rpm, absorb for 3h, filter, and detect the lipase content of the filtrate by the method of Example 1, The immobilization rate was calculated to be 92.4%; the filter cake was vacuum-dried at 40° C. to a water content of 3.1% to obtain 41.2 g of immobilized lipase.

2. Synthesis of sucrose-6-ethyl ester

Dimethyl sulfoxide and tert-amyl alcohol were mixed at a volume concentration of 30%: 70% to form a compound solvent of 1000ml, 35 grams of sucrose was added, and the sucrose was fully dissolved in the compound solvent by stirring, and 30 grams of immobilized lipase prepared in step 1 was added. Then add 250 grams of vinyl acetate, mix evenly, 30 ℃ water bath shaker speed 160r/min, reaction time 16h, filter to separate the immobilized enzyme, vacuum distillation to recover the solvent, to obtain 18.7g of sucrose ethyl ester product. The method of Example 1 was used for liquid chromatographic analysis. There were three parallel samples in each group, the esterification rate was 51.4%, and the 6-position esterification selectivity of sucrose was 86.8%.

Comparative Example 3:

1. Immobilization of lipase

Dissolve 20 grams of Aspergillus niger lipase (Aspergillus niger lipase, produced by Nanning Pangbo Biological Engineering Co., Ltd., 300,000 units/g) in 5000 ml of deionized water, stir to dissolve the enzyme, and filter to remove insoluble components. 21.8 g of disodium hydrogen phosphate dodecahydrate and 15.3 g of sodium dihydrogen phosphate were added, and stirred evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer. Add 50 grams of D3520 macroporous resin (Nankai University Chemical Plant) pretreated by the method of Example 1, keep at 30°C, keep stirring at 90 rpm, absorb for 3h, filter, and detect the lipase content of the filtrate by the method of Example 1, The immobilization rate was calculated to be 92.4%; the filter cake was vacuum-dried at 40° C. to a water content of 3.1% to obtain 41.2 g of immobilized lipase.

2. Synthesis of sucrose-6-ethyl ester

Tetrahydrofuran and tert-amyl alcohol were mixed with a volume concentration of 30%: 70% to form 1000ml of a composite solvent, 25 g of sucrose was added, and the sucrose was fully dissolved in the composite solvent by fully stirring, and 25 g of the immobilized lipase prepared in step 1 was added, and then acetic acid was added. 250 g of vinyl ester, mixed evenly, 30°C water bath shaker rotating speed 160 r/min, reaction time 16 h, filtered to separate the immobilized enzyme, and the solvent was recovered by vacuum distillation to obtain 9.6 g of sucrose ethyl ester product. The method of Example 1 was used for liquid chromatographic analysis, three parallel samples in each group, the esterification rate was 36.8%, and the 6-position esterification selectivity of sucrose was 65.7%.

Claims (9)

1. a method for enzymatic synthesis of sucrose-6-ethyl ester in an organic solvent, it is characterized in that the method is: sucrose is added in the organic solvent, then add immobilized lipase and vinyl acetate, mix homogeneously, at 30 -40°C water bath, shaking table rotating speed 120-180r/min for 12-20h, filter, filter cake to recover immobilized lipase, filtrate vacuum distillation to recover solvent to obtain sucrose-6-ethyl ester product; the organic solvent is made of N,N-dimethylformamide is mixed with tert-amyl alcohol or tert-butanol at a volume concentration of 55-30%: 45-70%, and the total amount is 100%; the immobilized lipase is made of lipase using large Pore resin adsorption and fixation. 2. the method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent as claimed in claim 1, it is characterized in that described organic solvent is by N,N-dimethylformamide and tertiary amyl alcohol with volume concentration 55～ 30%: 45～70% mixed, the total amount is 100%. 3. the method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent as claimed in claim 1, is characterized in that described organic solvent volume add-on is 125-330ml/g in sucrose weight; Described sucrose and immobilized The mass ratio of lipase is 1:0.2-1.0; the mass ratio of sucrose and vinyl acetate is 1:1-5. 4. the method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent as claimed in claim 1 is characterized in that described lipase enzyme activity is 10-300,000 units/gram. 5. the method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent as claimed in claim 4 is characterized in that lipase is Aspergillus niger lipase or Candida lipase. 6. The method for enzymatically synthesizing sucrose-6-ethyl ester in an organic solvent according to claim 1, wherein the macroporous resin types include D4006 type, D3520 type, and D1300 type. 7. the method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent as claimed in claim 1, it is characterized in that described immobilized lipase is prepared as follows: lipase is dissolved in deionized water, adds dodecahydrate Disodium hydrogen phosphate and sodium dihydrogen phosphate, stir evenly to form an enzyme solution of 0.05mol/L pH7.4 phosphate buffer, add macroporous resin, keep at 20-35 ℃, stir at 80-100 r/min, Adsorb for 1-3 hours, filter, and vacuum dry the filter cake to a water content of 3-5% to obtain immobilized lipase. 8. the method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent as claimed in claim 7, it is characterized in that described deionized water volume consumption is 150-500ml/g by lipase weight; The mass ratios of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate are 1:0.5-1.5 and 1:1:0.5-1.0 respectively; the weight ratio of the lipase to the macroporous resin is 1:2-4. 9. the method for enzymatic synthesis of sucrose-6-ethyl ester in organic solvent as claimed in claim 7, it is characterized in that described macroporous resin is pretreated before adding enzyme liquid, and described pretreatment method is: Add 10 times the mass of the 95% ethanol aqueous solution by volume to the pore resin, shake and wash for 24 hours at room temperature and 150 r/min, and replace the 95% ethanol aqueous solution twice in the middle. After the shaking and washing, use twice the mass each time. Washed with distilled water five times, suction filtered, and the filter cake is the macroporous resin that has completed the pretreatment.

Images